Background: Exposure to the particulate matter produced in underground railway systems is arousing increasing scientific interest because of its health effects. The aim of our study was to evaluate the airborne concentrations of PM10 and three sub-fractions of PM2.5 in an underground railway system environment in proximity to platforms and in underground commercial areas within the system, and to compare these with the outdoor airborne concentrations. We also evaluated the metal components, the cytotoxic properties of the various fractions of particulate matter (PM) and their capacity to induce oxidative stress. Method: We collected the coarse fraction (5–10 µm) and the fine fractions (1–2.5 µm; 0.5–1 µm; 0.25–0.5 µm). Chemical characterisation was determined by means of spectrometry. Cytotoxicity and oxidative stress were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Reactive Oxygen Species (ROS) assessment. Results: The concentrations of both PM10 and PM2.5 proved to be similar at the three sampling sites. Iron and other transition metals displayed a greater concentration at the subway platform than at the other two sites. The 2.5–10 µm and 1–2.5 µm fractions of PM from all three sampling sites determined a greater increase in ROS; the intensity of oxidative stress progressively declined as particle diameter diminished. Moreover, ROS concentrations were correlated with the concentrations of some transition metals, namely Mn, Cr, Ti, Fe, Cu, Zn, Ni and Mo. All particulate matter fractions displayed lower or similar ROS values between platform level and the outdoor air. Conclusions: The present study revealed that the underground railway environment at platform level, although containing higher concentrations of some particularly reactive metallic species, did not display higher cytotoxicity and oxidative stress levels than the outdoor air.